Project Summary From a siRNA screen performed at Vanderbilt, replication protein A (RPA) was identified as a novel target for triple negative breast cancer (TNBC). In the screen, knockdown of RPA resulted in attenuated cell growth of all 19 TNBC cell lines tested. RPA is a heterotrimeric single-stranded (ss) DNA-binding protein composed of 70, 32, and 14 kDa subunits and is essential for eukaryotic DNA replication, damage response, and repair. RPA functions as a scaffold upon which DNA processing proteins assemble and initiate DNA damage response pathways. Concurrently, RPA serves as a protective layer for ssDNA by preventing formation of aberrant DNA structures at replication foci. During DNA processing, the A, B, and C domains of the RPA70 subunit are responsible for binding ssDNA. The N-terminal domain of the RPA70 subunit interacts with a variety of proteins, such as p53, RAD9, ATRIP, and Mre11, that mediate DNA damage response pathways. Interferring with the action of RPA70N by blocking the ability of the 70N subunit to recruit DNA processing proteins should have an effect on several critical DNA damage response pathways, such as those mediated by p53 and the ATR pathway. Further, this inhibition should produce a more selective effect than the siRNA screen, as the ssDNA binding functions of RPA should not be affected. The inhibition of protein-protein interactions is difficult using traditional methods. In order to selectively inhibit RPA70N-mediated protein-protein interactions, we propose to discover both a potent stapled helix peptide probe and a small molecule probe. We have identified a candidate stapled helix peptide probe that binds tightly to RPA70N and penetrates cells. In addition, we have identified compounds that bind to two adjacent sites on RPA70N using an NMR-based fragment screen, and have also identified molecules that span the two binding sites using a high throughput screen. Structure-based optimizations have produced multiple lead series of compounds that bind to RPA70N with sub-micromolar affinities. In this proposal, our goal is to confirm the suitability of the peptide as a cellular probe and to obtain potent small molecule probes that are suitable for testing in a wide panel of cell lines. We will use these probes to test the hypothesis that selective inhibition of RPA70N-mediated protein-protein interactions is a valid approach for cancer therapy. We will further define the molecular determinants of activity for these inhibitors and determine the functional signifance of RPA70N inhibition. If successful, we will have identified a promising strategy for targeting cancer. In addition, we will have produced useful and potent probe molecules that could not only be used to enable further understanding of RPA biology, but also be further optimized into a drug for the treatment of breast cancer and other tumor types. !